Acanthamoeba castellanii contains three single-headed myosins. Myosin IA has a heavy chain of 130,000 and light chains of 17,000 and 14,000 daltons; IB has a heavy chain of 125,000 and light chains of 27,000 and 14,000 daltons; IC has a heavy chain of 130,000 and light chains of 20,000, 17,000 and 14,000 daltons. Removal of the 20,000-dalton peptide from IC converts it to IA. The 20,000-dalton peptide is a regulatory protein that inhibits the Ca2 ion - and Mg2 ion -ATPase activities, and, less completely, the (K ion, EDTA)- and actin-activated MG2 ion -ATPase activities on myosin IA. All of the enzymatic activities of myosin IB can be recovered in the heavy chain free of light chains. Acanthamoeba also contains a two-headed enzyme, myosin II, consisting of two heavy chains of 17,000 and two pairs of light chains of 17,500 and 17,000 daltons. Trypsin digestion converts myosin II into a two-headed heavy meromyosin and, eventually, a single-headed subfragment-1 that retain the unique enzymatic properties of myosin II. Peptide maps of cleavage products formed by cyanogen bromide, cyanylation and protease digestion confirm that myosin IA and myosin IC are identical and establish that myosin II differs from all three myosins I. Acanthamoeba castellanii must, therefore, express at least two, and probably three, myosin genes.